PROJECT SUMMARY The marked increase in the frequency and spread of multidrug-resistant (MDR) Gram-negative bacterial (GNB) infections has created an urgent need for alternative therapeutic approaches. Given that the mechanisms to evade antibiotics is evolving more rapidly than the development of new antibiotics, novel approaches in a ?post-antibiotic? era are needed. Immunotherapeutic approaches, such as vaccines, are less prone to the emergence of resistant organisms and avoid the possibility of immune compromise that complicate strategies that target the host response. Vaccine-induced antibodies against bacteria, including those directed against the conserved LPS core (lipooligosaccharide, LOS) region of GNB are highly effective and protective against a broad range of clinically relevant GNB in both animal models of infection and in human studies. Previously, we developed a subunit LOS-based vaccine, an innovative formulation of the earlier heat-killed E. coli O111, J5- mutant vaccine. The purified LOS required chemical detoxification to reduce reactogenicity, and non-covalent complexing with group B N. meningitides (Nm) outer membrane protein (OMP) to improve immunogenicity. The resulting J5dLOS/OMP vaccine was well-tolerated in two Phase 1 clinical trials and highly immunogenic when administered with CPG 7909 adjuvant. We now propose the development of a next generation J5 LOS vaccine that will be genetically detoxified and have intrinsic TLR4-adjuvant activity while retaining the conserved core LOS epitope. Through the use of bacterial enzymatic combinatorial chemistry (BECC), the Ernst laboratory has pioneered the production of lipid A mimetics with reduced endotoxicity but which retain monophosphoryl lipid A-like adjuvant activity. In Specific Aim 1, we will generate a series of rationally- designed targeted modifications in the lipid A portion of E. coli J5 mutant, prepare LOS from each modified isolate, characterize the structure and assess their endotoxicity in HEKT293-Blue cells. In Specific Aim 2, we will compare the immunogenicity of the genetically-detoxified LOSs to that of the chemically-detoxified J5 LOS with and without complexing to recombinant OMP. We will select those LOSs that are most immunogenic and with reduced endotoxicity for evaluation of protective activity in murine models of lethal peritonitis and pneumonia caused by an MDR strain of Klebsiella pneumoniae. At the conclusion of these studies, we will evaluate if a genetically-detoxified, intrinsically-adjuvanted J5 LOS vaccine is equivalent or superior to the current J5dLPS/OMP vaccine already shown to induce a robust antibody response in healthy human subjects. A successful vaccine could be used to generate antibodies for the treatment of a broad range of MDR GNB infections, a huge, unmet medical need, either alone or in synergistic combination with antibiotics, or for the prevention of infection in patients at high risk of severe GNB infections.